The proposal will focus on the role of physiological beta cell apoptosis and the role of tumor necrosis factor alpha (TNFalpha) in the development of type 1 diabetes (T1DM). Type 1 diabetes is an autoimmune disease resulting from specific destruction of the beta-islet cells of the pancreas. Several reports have shown that apoptosis of beta-cells may play a crucial role at specific time points during disease progression. The proposed studies will test whether diabetes can be decreased or prevented by blocking apoptosis in the critical 17-21 day neonatal period. These experiments will explore the in vivo effects of quantitatively and temporally controlled expression of anti-apoptotic protein in beta-islet cells using a tetracycline-regulated gene expression (Tet-on) system with the rat insulin promoter RIP-7. Separate experiments will study the effect of neonatal TNF and anti-TNF therapy on the activation, or prevention of activation of islet and regional lymph node dendritic cells. Studies by the mentor, Dr. McDevitt, and others have shown that treatment in the neonatal period with TNFalpha, a potent apoptotic inducer in several model systems, leads to an earlier onset and increased incidence of T 1DM in NOD mice. Further, neonatal anti-TNF treatment completely prevents T1DM in NOD mice. The molecular mechanisms are not well understood. Paradoxically, chronic TNFalpha exposure in adult mice decreases T cell receptor signaling, while adult TNFalpha blockade appears to increase T cell effector function. The specific aims include: (1) Generation of tetracycline-regulated gene expression (Tet-on) system; this will use RIP7-rtTA, TRE-myc-CrmA and TRE-myc-Bclxl constructs; (2) Generation and characterization of RIP-7-rtTA, TRE-myc-CrmA, and TRE-myc-Bclxl transgenic NOD mice; (3) Characterization of neonatal and adult in vivo TNF and anti-TNF exposure in NOD mice; (4) Analysis of the effects of chronic in vivo TNFalpha and anti-TNFalpha exposure on the TNFalpha and T cell receptor signaling cascades in BDC 2.5 T cell receptor transgenic mice, by gene expression profiling on cDNA microarrays and by specific immunoblotting with anti-phosphotyrosine and anti-phosphoserine. These studies will aid in understanding the molecular mechanism ofapoptosis and the role of TNF and anti-TNF in the development of T1DM, as well as aid in understanding the relationship between the TNF and TCR signaling cascades.